EP1491634A1 - Feedback resistente Acetohydroxysäure-Synthetase Mutanten - Google Patents

Feedback resistente Acetohydroxysäure-Synthetase Mutanten Download PDF

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Publication number
EP1491634A1
EP1491634A1 EP20030014640 EP03014640A EP1491634A1 EP 1491634 A1 EP1491634 A1 EP 1491634A1 EP 20030014640 EP20030014640 EP 20030014640 EP 03014640 A EP03014640 A EP 03014640A EP 1491634 A1 EP1491634 A1 EP 1491634A1
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Prior art keywords
nucleic acid
polypeptide
seq
ahas
activity
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EP20030014640
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English (en)
French (fr)
Inventor
Miroslav Patek
Veronika Elisakova
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Evonik Operations GmbH
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Degussa GmbH
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Priority to EP20030014640 priority Critical patent/EP1491634A1/de
Priority to DE602004024394T priority patent/DE602004024394D1/de
Priority to JP2006515843A priority patent/JP2007506407A/ja
Priority to PCT/EP2004/006157 priority patent/WO2005003357A1/en
Priority to US10/561,906 priority patent/US7585959B2/en
Priority to KR1020057024680A priority patent/KR20060024437A/ko
Priority to CNB2004800179270A priority patent/CN100523197C/zh
Priority to AT04739686T priority patent/ATE450611T1/de
Priority to RU2006102024/13A priority patent/RU2325439C2/ru
Priority to EP04739686A priority patent/EP1636366B1/de
Priority to BRPI0411975-4A priority patent/BRPI0411975A/pt
Publication of EP1491634A1 publication Critical patent/EP1491634A1/de
Priority to ZA200510448A priority patent/ZA200510448B/xx
Priority to US12/496,475 priority patent/US20100086966A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present invention is directed to specific nucleic acids and polypeptides coded by these nucleic acids as well as their application.
  • the polypeptides of the present invention serve to improve the production of branched-chain amino acids by fermentation.
  • the present invention provides nucleotide sequences coding for acetohydroxy acid synthetase (AHAS) mutants, the mutated enzymes themselves and a process for the fermentative production of branched-chain amino acids using these enzymes in specific hosts in which genes which code for the modified acetohydroxy acid synthetase (AHAS) are expressed.
  • AHAS acetohydroxy acid synthetase
  • amino acids may be produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum . Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the micro-organism itself.
  • strains are obtained which are resistant to antimetabolites, such as for example the isoleucine analogue isoleucine hydroxyamate (Kisumi M, Komatsubara S, Sugiura, M, Chibata I (1972) Journal of Bacteriology 110: 761-763), the valine analogue 2-thiazolealanine (Tsuchida T, Yoshinanga F, Kubota K, Momose H (1975) Agricultural and Biological Chemistry, Japan 39: 1319-1322) or the leucine analogue ⁇ -aminobutyrates (Ambe-Ono Y, Sato K, Totsuka K, Yoshihara Y, Nakamori S (1996) Bioscience Biotechnology Biochemistry 60: 1386-1387) or which are auxotrophic for regulatorily significant metabolites and produce e.g.
  • antimetabolites such as for example the isoleucine analogue isoleucine hydroxyamate (Kisumi M, Komatsubara S, Sugiura
  • L-isoleucine L-valine and L-leucine are used in pharmaceutical industry, in human medicine and in animal nutrition.
  • One of the key enzymes of the synthesis of all three amino acids in bacteria is the acetohydroxy acid synthetase (AHAS). It catalyses two reactions giving rise to precursors of the three amino acids.
  • AHAS acetohydroxy acid synthetase
  • AHAS In valine and leucine biosynthesis pathway, the substrate for AHAS is pyruvate. AHAS catalyses the decarboxylation of pyruvate and its condensation with the second molecule of pyruvate to produce acetolactate. In the isoleucine pathway, AHAS catalyses reaction of pyruvate and 2-ketobutyrate producing acetohydroxy butyrate. In Escherichia coli strains, as much as three AHAS isoenzymes exist. Activity of the isoenzymes is inhibited by combinations of amino acids, from which the inhibition by valine is the strongest (De Felice, M., Levinthal, M., Iaccarino, M., Guardiola, J., 1979.
  • AHAS I coded by the genes ilvBN
  • AHAS II coded by ilvGM
  • AHAS III coded by ilvIH is inhibited by valine and isoleucine
  • the enzyme consists of 2 subunits.
  • AHAS I and AHAS III the small regulatory subunits coded by the genes ilvN and ilvH , respectively, are responsible for the inhibition.
  • ilvBNC acetohydroxy acid synthetase in Corynebacterium glutamicum during fermentation of alfa-ketobutyrate to L-isoleucine. Appl Microbiol Biotechnol 25, 346-351). Expression of the gene cluster ilvBNC is also regulated by these three amino acids through the transcriptional attenuation (Morbach, S., Junger, C., Sahm, H., Eggeling, L., 2000. Attenuation control of ilvBNC in Corynebacterium glutamicum : evidence of leader peptide formation without the presence of a ribosome binding site. J Biosci Bioeng 90, 501-507).
  • the object of the present invention was to provide a modified acetohydroxy acid synthetase (AHAS).
  • AHAS modified acetohydroxy acid synthetase
  • the AHAS of the present invention shall be less prone to inhibition by amino acids just produced.
  • Claim 1 is directed to specific nucleic acids which code for a polypeptide comprising envisaged features.
  • Claim 2 embraces the polypeptides themselves.
  • Claim 3 and 4 disclose hosts comprising the nucleic acids of the invention or special primers or probes for their production via PCR.
  • claim 5 specifies a process for the production of further improved polypeptides of the inventions, whereas claim 6 protects the thus produced polypeptides and nucleic acids, respectively.
  • Claim 7 and 8 are directed to special uses and claim 9 embraces a process for the production of amino acids.
  • claim 10 and 11 provide special vectors and micro-organisms.
  • AHAS acetohydroxy acid synthetase
  • Suitable methods of mutagenesis are all the methods available for this purpose to a person skilled in the art. In particular these include saturation mutagenesis, random mutagenesis, in vitro recombination methods and site-directed mutagenesis (Eigen, M. and Gardiner, W., Evolutionary molecular engineering based on RNA replication, Pure Appl. Chem. 1984, 56 , 967-978; Chen, K. and Arnold, F., Enzyme engineering for non-aqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media.
  • nucleic acid sequences acting as probes or primers have at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleic acids in common with those of the invention. Nucleic acid sequences having a length of at least 40 or 50 base pairs are also suitable.
  • the invention also provides rec-polypeptides or nucleic acid sequences coding for these which are obtainable by a process like the one just described. Preparation of the nucleic acid sequences required to produce the improved rec-polypeptides and their expression in hosts is described supra and accordingly applies here.
  • polypeptides and improved rec-polypeptides according to the invention are preferably used to prepare enantiomer-enriched branched-chain amino acids, more preferably valine, leucine and isoleucine.
  • nucleic acid sequences and improved nucleic acid sequences may preferentially be used to prepare an branched-chain amino acid producing micro-organism.
  • a next development of the invention reflects a process for the production of branched-chain amino acids with utilises a polypeptide of the invention.
  • vectors pECKA Fig. 1
  • pECKA/ilvBNC Fig. 2
  • modified micro-organisms like DSM15652, DSM15561 or DSM15650 are enclosed in present invention. They were deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, according to the Budapest Treaty on June 04, 2003.
  • the shuttle vector Escherichia coli - Corynebacterium glutamicum was constructed. First recognition site for the restriction enzyme Bgl II was removed from the vector pK19. Then, Hind III/ Hind II fragment (2.7 kb) of the plasmid pBL1 from Brevibacterium lactofermentum was cloned into Nhe I site of pK19.
  • the resulting plasmid vector pECKA (5.4 kb) replicates in Escherichia coli and Corynebacterium glutamicum , provides 7 unique cloning sites, kanamycin resistance marker and ⁇ -complementation of ⁇ -galactosidase for cloning in E. coli .
  • the Chromosomal fragment Ssp I/ Eco RI (5.7 kb) (with Ssp I+ Bam HI ends) carrying the ilvBNC operon was cloned into the Hind II+ Bam HI-digested vector pECKA to create pECKA ilvBNC (11.1 kb).
  • the natural Sca I/ Bgl II fragment of ilvBNC operon (770 bp) was exchanged with the same fragment containing 3 to 5 base alterations constructed by site-directed mutagenesis.
  • the target for site-directed mutagenesis was the conserved domain of the regulatory subunit coded by ilvN near the N terminus. Mutations were designed by PCR according to the sequences of the Escherichia coli and Streptomyces cinnamonensis AHAS mutants. Mutations were detected by sequencing.
  • Plasmid DNA was isolated from Escherichia coli and the strain Corynebacterium glutamicum ATCC13032 ⁇ ilvN was transformed with the plasmids pECKA ilvBNC (WT), pECKA ilvBNC (M8) and pECKA ilvBNC (M13). The decrease of inhibition of AHAS by branched-chain amino acids was demonstrated.
  • Isolated means separated from its natural environment.
  • Optically enriched (enantiomerically enriched, enantiomer enriched) compounds in the context of this invention is understood to mean the presence of >50 mol% of one optical antipode mixed with the other.
  • nucleic acid sequences is intended to include all types of single-strand or double-strand DNA and also RNA or mixtures of the same.
  • an improvement in activity and/or selectivity and/or stability means, according to the invention, that the polypeptides are more active and/or more selective and are more stable under the reaction conditions used.
  • activity and stability of enzymes for industrial application should naturally be as high as possible, with regard to the selectivity an improvement is referred to either when either the substrate selectivity decreases or the enantioselectivity of the enzymes increases.
  • the same definition applies mutatis mutandis.
  • the claimed protein sequences and nucleic acid sequences also include, according to the invention, those sequences which have a homology (excluding natural degeneration) of greater than 91 %, preferably greater than 92 %, 93 % or 94 %, more preferably greater than 95 % or 96 % and particularly preferably greater than 97 %, 98 % or 99 % to one of these sequences, provided the mode of action or purpose of such a sequence is retained.
  • nucleic acid sequences which code for polypeptides includes all sequences which appear to be possible, in accordance with degeneration of the genetic code.
  • the shuttle vector replicating in Escherichia coli and Corynebacterium glutamicum was constructed.
  • recognition site for the restriction enzyme Bgl II was removed from the vector pK19 (Pridmore, R. D., 1987. New and versatile cloning vectors with kanamycin-resistance marker. Gene 56, 309-312).
  • the plasmid pK19 was digested by Bgl II, blunt-ended by Klenow enzyme and religated. After ligation, E.
  • coli DH5 ⁇ cells were transformed with the ligation mixture and transformants containing the resulting plasmid pK19B were selected on agar plates containing kanamycin (20 mg/l).
  • the removal of the Bgl II site in pK19B was confirmed by the treatment of the isolated plasmid molecule with Bgl II. (This removal has permitted later subcloning of the fragment carrying the ilvN gene into the newly constructed vector pECKA.)
  • Hind III/ Hind II fragment (2.7 kb) of the plasmid pBL1 from Brevibacterium lactofermentum blunt-ended by the Klenow enzyme was cloned into the blunt-ended Nhe I site of pK19B.
  • the resulting plasmid vector pECKA (5.4 kb) replicates in Escherichia coli and Corynebacterium glutamicum , provides 7 unique cloning sites ( Hind II, Sal I, Bam HI, SmaI, Ava I, Kpn I, Sac I) kanamycin resistance marker and ⁇ -complementation of ⁇ -galactosidase for cloning in E. coli . Its restriction and genetic map is shown in Fig. 1.
  • the 5.7-kb fragment of C. glutamicum chromosome carrying the ilvBNC operon was obtained by digestion of the plasmid pKK5 (Keilhauer, C., Eggeling, L., Sahm, H., 1993. Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J. Bacteriol. 175, 5595-5603) with the restriction enzymes Ssp I and Bam HI. The fragment was ligated with the Hind II+ Bam HI-digested vector pECKA and the ligation mixture was used for transformation of E. coli DH5 ⁇ .
  • the transformants were selected on the agar plates containing kanamycin (30 mg/l).
  • the structure of the resulting plasmid pECKA ilvBNC (11.1 kb) was confirmed by restriction analysis.
  • the restriction and genetic map of the plasmid pECKA ilvBNC is shown in Fig. 2.
  • the known amino acid sequence of the regulatory subunit of AHAS coded by the C. glutamicum ilvN gene was aligned with the known amino acid sequences of regulatory subunits of AHAS from Streptomyces cinnamonensis (GenBank accession number AF175526) and from Escherichia coli (GenBank accession number AE016769, section 15 of the complete genome).
  • Streptomyces cinnamonensis GenBank accession number AF175526
  • Escherichia coli GenBank accession number AE016769, section 15 of the complete genome.
  • Several mutations of Escherichia coli and Streptomyces cinnamonensis conferring resistance to valine were described (Vyazmensky, M., Sella, C., Barak, Z., Chipman, D. M., 1996.
  • ILVNM1 degenerated oligonucleotide primer ILVNM1 (SEQ. ID NO: 7) for site-directed mutagenesis of the ilvN gene of C. glutamicum .
  • This primer may introduce mutations into the ilvN gene at the positions of the nucleotide triplets corresponding to the amino acids glycine, isoleucine and isoleucine at positions 20 to 22 in C. glutamicum AHAS regulatory subunit:
  • nucleotides altered comparing to the sequence of the wild type, are shown in bold face. There are two degenerated positions, within triplets 20 and 22 (G or A and A or T, respectively).
  • Second PCR Using primers MILVNH - MILVND and template fragments A + B (mixed in a molar ratio 1:1), a mixture of fragment C (803 bp) with mutation in Bgl II site and fragment D (803 bp) with mutations in the ilvN gene were amplified. This mixture was digested by Sca I and Bgl II and the resulting fragments were isolated from the agarose gel. The plasmid pECKA ilvBNC was digested by the same enzymes providing fragments of 766 bp and 10334 bp and the larger fragment was also isolated from the gel. The isolated fragments were mixed and ligated. The cells of E.
  • coli DH5 ⁇ were transformed by the ligation mixture and transformants were selected on the plates with kanamycin (30 mg/l). In this way, a natural Sca I/ Bcl II chromosomal fragment (766 bp) in the plasmid pECKAilvBNC was exchanged for the same fragment in which ilvN can contain 3 to 5 altered nucleotides.
  • Plasmid DNA from the obtained E. coli DH5 ⁇ clones was isolated and sequenced using the primer SILVNH and automatic sequencer Vistra (Amersham).
  • Plasmid DNA was isolated from Escherichia coli and the strain Corynebacterium glutamicum ATCC13032 ⁇ ilvN was transformed with the plasmids pECKA ilvBNC (WT), pECKA ilvBNC (M8), pECKA ilvBNC (M11) and pECKA ilvBNC (M13) using the electroporation method (Liebl, W., Bayerl, A., Schein, B., Stillner, U., Schleifer, K. H., 1989. High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol. Lett. 53, 299-303).
  • Transformants were selected on the plates with kanamycin (30 mg/l).
  • the assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex.
  • AHAS activity Strain/plasmid Specific AHAS activity (nmol acetoin min -1 mg -1 of protein) C. glutamicum ATCC13032 33.7 ⁇ 10 C. glutamicum ATCC13032 ⁇ ilvN 0.43 C. glutamicum ATCC13032 ⁇ ilvN / pECKAilvBNC (WT) 110 ⁇ 40 C. glutamicum ATCC13032 ⁇ ilvN /pECKA ilvBNC (M8) 31.1 ⁇ 0.9 C .
EP20030014640 2003-06-26 2003-06-26 Feedback resistente Acetohydroxysäure-Synthetase Mutanten Withdrawn EP1491634A1 (de)

Priority Applications (13)

Application Number Priority Date Filing Date Title
EP20030014640 EP1491634A1 (de) 2003-06-26 2003-06-26 Feedback resistente Acetohydroxysäure-Synthetase Mutanten
AT04739686T ATE450611T1 (de) 2003-06-26 2004-06-08 Feedback-resistente acetohydroxysäure-synthetase- mutanten
RU2006102024/13A RU2325439C2 (ru) 2003-06-26 2004-06-08 Мутанты синтетазы ацетооксикислот, устойчивые к обратной связи
PCT/EP2004/006157 WO2005003357A1 (en) 2003-06-26 2004-06-08 Feedback resistant acetohydroxy acid synthethase mutants
US10/561,906 US7585959B2 (en) 2003-06-26 2004-06-08 Feedback resistant acetohydroxy acid synthethase mutants
KR1020057024680A KR20060024437A (ko) 2003-06-26 2004-06-08 피드백 내성 아세토하이드록시산 합성효소 돌연변이체
CNB2004800179270A CN100523197C (zh) 2003-06-26 2004-06-08 反馈抗性乙酰羟酸合成酶突变体
DE602004024394T DE602004024394D1 (en) 2003-06-26 2004-06-08 Feedback-resistente acetohydroxysäure-synthetase-mutanten
JP2006515843A JP2007506407A (ja) 2003-06-26 2004-06-08 フィードバック耐性アセトヒドロキシ酸シンターゼ変異体
EP04739686A EP1636366B1 (de) 2003-06-26 2004-06-08 Feedback-resistente acetohydroxysäure-synthetase-mutanten
BRPI0411975-4A BRPI0411975A (pt) 2003-06-26 2004-06-08 mutantes da acetohidróxi ácido sintetase resistentes à retroalimentação
ZA200510448A ZA200510448B (en) 2003-06-26 2005-12-22 Feedback resistant acetohydroxy acid synthetase mutants
US12/496,475 US20100086966A1 (en) 2003-06-26 2009-07-01 Feedback resistant acetohydroxy acid synthethase mutants

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EP20030014640 EP1491634A1 (de) 2003-06-26 2003-06-26 Feedback resistente Acetohydroxysäure-Synthetase Mutanten

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EP20030014640 Withdrawn EP1491634A1 (de) 2003-06-26 2003-06-26 Feedback resistente Acetohydroxysäure-Synthetase Mutanten
EP04739686A Not-in-force EP1636366B1 (de) 2003-06-26 2004-06-08 Feedback-resistente acetohydroxysäure-synthetase-mutanten

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US (2) US7585959B2 (de)
EP (2) EP1491634A1 (de)
JP (1) JP2007506407A (de)
KR (1) KR20060024437A (de)
CN (1) CN100523197C (de)
AT (1) ATE450611T1 (de)
BR (1) BRPI0411975A (de)
DE (1) DE602004024394D1 (de)
RU (1) RU2325439C2 (de)
WO (1) WO2005003357A1 (de)
ZA (1) ZA200510448B (de)

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EP1942183A1 (de) * 2006-09-13 2008-07-09 Ajinomoto Co., Inc. Mutante Acetolactat-Snythase und Verfahren zur Herstellung verzweigter L-Aminosäuren
DE102011118019A1 (de) 2011-06-28 2013-01-03 Evonik Degussa Gmbh Varianten des Promotors des für die Glyzerinaldehyd-3-phosphat-Dehydrogenase kodierenden gap-Gens
EP2811028A1 (de) 2013-06-03 2014-12-10 Evonik Industries AG Verfahren zur Herstellung von L-Leucin, L-Valin, L-Isoleucin, alpha-Ketoisovalerat, alpha-Keto-beta-Methylvalerat oder alpha-Ketoisocaproat unter Verwendung rekombinanter Corynebakterien enthaltend das durch Propionat induzierbare ilvBN-Operon

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WO2012014228A1 (en) 2010-07-28 2012-02-02 Abhishek Narain Singh A method to by-pass allosteric domain activity of an enzyme so as to alter its feedback or feed-forward inhibition or activation
KR101720836B1 (ko) 2014-08-05 2017-04-03 씨제이제일제당 (주) 피드백 저항성 아세토하이드록시산 신타아제 변이체 및 이를 이용한 l-발린의 생산방법
CN105886431B (zh) * 2016-04-27 2019-05-10 天津科技大学 一株谷氨酸棒状杆菌及其高产异亮氨酸的方法
WO2017194696A1 (en) 2016-05-12 2017-11-16 Danmarks Tekniske Universitet Bacterial cells with improved tolerance to isobutyric acid
KR101996129B1 (ko) 2017-07-11 2019-07-04 씨제이제일제당 (주) 아세토하이드록시산 신타아제 변이체, 이를 포함하는 미생물 또는 이를 이용하는 l-분지쇄 아미노산 생산 방법
US11021697B2 (en) 2017-07-11 2021-06-01 Cj Cheiljedang Corporation Acetohydroxy acid synthase variant, microorganism comprising the same, and method of producing L-branched-chain amino acid using the same
CA3119088A1 (en) * 2018-12-12 2020-06-18 Utilization Of Carbon Dioxide Institute Co., Ltd. A recombinant hydrogenophilus bacterium with enhanced ability to produce valine
CN110229797B (zh) * 2019-06-05 2020-08-28 天津科技大学 一种乙酰羟酸合成酶及其应用
KR102147381B1 (ko) * 2019-11-22 2020-08-24 씨제이제일제당 주식회사 아세토하이드록시산 신타제 신규 변이체 및 이를 포함하는 미생물
KR20230045989A (ko) * 2021-09-29 2023-04-05 씨제이제일제당 (주) 신규한 아세토하이드록시산 신테아제 변이체 및 이를 이용한 l-이소류신 생산방법
KR20230045990A (ko) * 2021-09-29 2023-04-05 씨제이제일제당 (주) 신규한 아세토하이드록시산 신테아제 변이체 및 이를 이용한 l-이소류신 생산방법

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EP2811028A1 (de) 2013-06-03 2014-12-10 Evonik Industries AG Verfahren zur Herstellung von L-Leucin, L-Valin, L-Isoleucin, alpha-Ketoisovalerat, alpha-Keto-beta-Methylvalerat oder alpha-Ketoisocaproat unter Verwendung rekombinanter Corynebakterien enthaltend das durch Propionat induzierbare ilvBN-Operon
US10113190B2 (en) 2013-06-03 2018-10-30 Evonik Degussa Gmbh Method for producing L-leucine, L-valine, L-isoleucine, α-ketoisovalerate, α-keto-beta-methylvalerate, or α-ketoisocaproate using recombinant Corynebacteria that contain the ilvBN operon which can be induced by propionate

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US7585959B2 (en) 2009-09-08
WO2005003357A1 (en) 2005-01-13
US20100086966A1 (en) 2010-04-08
EP1636366B1 (de) 2009-12-02
CN1813065A (zh) 2006-08-02
ATE450611T1 (de) 2009-12-15
RU2325439C2 (ru) 2008-05-27
CN100523197C (zh) 2009-08-05
DE602004024394D1 (en) 2010-01-14
RU2006102024A (ru) 2006-09-10
US20070292914A1 (en) 2007-12-20
ZA200510448B (en) 2006-12-27
KR20060024437A (ko) 2006-03-16
JP2007506407A (ja) 2007-03-22
BRPI0411975A (pt) 2006-08-29

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